The publications and other materials used herein to illuminate the background of the invention, and in particular, cases to provide additional details respecting the practice, are incorporated by reference.
Förster type of non-radiative dipole-dipole energy transfer (Förster (1948) Ann. Physik., 6, 55) takes place between two molecules in condition where their energies (emission of donor (D) with absorption of acceptor (A)) overlap and they are located at a suitable distance from each other. The energy transfer requires a proper orientation of the oscillations of the molecules. The energy transfer efficiency E is given in the equation E=1/(1+r6/R06), in which r means the distance between the donor and the acceptor, and R0 is a distance parameter characteristic of the donor-acceptor pair and the medium between them. The usable distance scale for FRET experiments using conventional donor—acceptor pairs is ˜10-100 Å.
Fluorescence resonance energy transfer (FRET), also called luminescence resonance energy transfer (LRET), has found extensive applications both in basic research and bioanalytical technologies as a platform for homogenous assays and as a spectroscopic ruler to measure distances in biomolecules. Ullman was the first to describe application of Förster type non-radiative energy transfer in homogenous bioanalytical assays based on antibody recognition reaction (Ullman, Scharzberg and Rubenstein (1967) J. Biol. Chem., 251: 4172) and a great number of suitable donor-acceptor probe pairs have since been developed and applied in immunoassays (for a review see I Hemmilä, Applications of Fluorescence in Immunoassays, Wiley, NY, 1991, chapter 8.3.4).
Time-resolved (TR) fluorometry (time resolution in time-domain at micro- or millisecond range) is an excellent measuring regime for homogenous assays because it can totally discriminate against ns-lifetime background fluorescence caused by organic compounds and light scattering. Suitable donors for TR-FRET measurements include among others lanthanide chelates (cryptates) and some other metal ligand complexes, which can have fluorescent lifetime in the micro-millisecond time region, and which therefore also allow the energy transfer to occur in micro-millisecond time region. This enables the time-resolved detection of the FRET-signal. Especially lanthanides and their fluorescent complexes have established a strong position as donors in TR-FRET measurements. Fluorescent lanthanide chelates have been used as energy donors already since 1978 by Stryer, Thomas and Meares (For example see Thomas et al. (1978) Proc. Natl. Acad. Sci., 75: 5746) and since a number of homogenous TR-FRET based assays have been described and patended (Mathis (1995) Clin. Chem., 41, 1391; Selvin et al. (1994) Proc. Natl. Acad. Sci., USA, 91, 10024) with their limitations and drawbacks.
Assay sensitivity has always been a critical parameter in bioassays. Very efficient way to measure a FRET based assay is to monitor the acceptor fluorescence instead of donor fluorescence. This is because in ideal circumstances the acceptor signal is specific only to the energy transfer (acceptors exited due to energy transfer) and the fluorescence signal is formed from only one fluorescent population, which makes the sensitive detection easier. Acceptor fluorescence monitoring also helps to avoid problems related to incomplete labeling of the FRET probes. In contrast the donor fluorescence basically always contains at least two different fluorescent populations: donors that participate in the energy transfer and free donors. Both populations emit at the same wavelength, which means that the change in the donor fluorescence signal is always measured against high background (free donors) in donor fluorescence channel.
Acceptor signal monitoring in FRET-assay has its own limitations, which are based on the energy transfer scheme of the probe pair and the fact that in most of cases the absorption of the acceptor overlaps with the emission of the donor (for non-overlapping energy transfer, see U.S. Pat. No. 5,998,146). Therefore in photo-absorption based excitation methods the acceptor molecules will be excited directly by the donor excitation light to a degree proportional to the intensity of the external light source. Also due to the spectral overlap the excited donor always emits background fluorescence to the acceptor emission channel in some extent. The fluorescence signal resulting from these sources to the acceptor emission channel is non-specific to the energy transfer and limits the sensitivity in acceptor emission based FRET assays.
Methods to overcome these limitations has been described and patented. The use of temporal discrimination (long lifetime donor, short lifetime acceptor; and time-resolution) to avoid the effect of directly excited acceptor molecules was first described by Morrison (Morrison (1988) Anal. Biochem., 174, 101; U.S. Pat. No. 4,822,733). He used organic donor-acceptor pair; pyrene donor (τ=400 ns) and phycoerythrin acceptor (τ=4 ns). The effect of donor background fluorescence in acceptor emission channel can be reduced using donors with narrow emission bands, which enables more efficient optical filtering of the acceptor fluorescence. For example Selvin et al. have described significant improvements to S/B-ratio using europium- and terbium chelates as donors in TR-FRET measurements (Selvin et al. (1994) J. Am. Chem Soc., 116, 6029-6030; Selvin et al. (1994) Proc. Natl. Acad. Sci, USA, 91, 10024-10028). Mathis et al. used lanthanide cryptates, which have low overall quantum yield, as donors in TR-FRET assay to reduce the background caused by free donors (U.S. Pat. No. 5,512,493). Nevertheless there still exist a need for more sensitive FRET-assays.
Methods for homogenous FRET based multianalyte detection, in which more than one analyte can be detected simultaneously from the same assay medium, have also been described. The TAQMAN® method (Lee et al. (1993) Nucl. Acids. Res., Vol 21, 16, 3761-3766) uses dual labeled oligonucleotide probes, in which both the fluorescent reporter dye and the quencher are attached in the same oligonucleotide and TAQ-DNA polymerase cleavage of the probe causes the signal change in the assay. In TAQMAN® dual-assay the donors have different emission wavelengths and the donor signals are resolved by spectrally means. Molecular Beacon method (Tyagi S. and Kramer F. R. (1996) Nat. Biotech., 14, 303-308, U.S. Pat. No. 5,925,517) uses also dual labeled oligonucleotide probes, in which both fluorescent reporter dye and quencher are attached in the same oligonucleotide. The signal change is based on the self-hybridization of the probe. When the probe is not bound to the target the donor fluorescence is quenched due to the self-hybridization of the probe, which brings the reporter dye and the quencher in close proximity. When bound to target the self hybridization is avoided and the donor fluorescence can be obtained. In dual molecular beacon assay the donors have different emission wavelengths and the different donor signals are resolved by spectrally means. Hemmilä et al. have described a TR-FRET based homogenous multianalyte detection, in which separate donor-acceptor pairs are used for different analytes (WO 98/15830). The method is not described in detail but the donors are claimed to be lanthanide chelates (Eu, Tb, Sm) and acceptors are claimed to be short lifetime fluorophores. One can assume that the signal separation in the method described by Hemmilä is based either on spectrally resolvable donor emissions and/or spectrally resolvable acceptor emissions.
The problem related to optical signal separation methods is that usually the emission spectra of the suitable acceptor fluorophores are broad. It is difficult to totally avoid so-called crosstalk between the dye signals, which means that one dye also emits a certain percent of its total emission at the wavelength of a second dye. In high sensitivity assay this kind of crosstalk decreases the detection limit of the assay and can lead to false positive results, especially, if the one of the detected compounds has a large excess compared to the other compounds. Spectral crosstalk can be corrected mathematically afterwards, but it makes the data handling more difficult. Neither one of the methods described above is suitable for simultaneous multianalyte detection using one and the same donor for all analytes. Method described by Hemmilä (WO 98/15830) allows the use of one donor and different acceptors in multilabel assay but because of the spectral dependence of the energy transfer the suitable acceptors for the same donor have to have quite similar optical properties. This makes it difficult to avoid optically the crosstalk of different acceptor signals.
Conventional homogenous TR-FRET bioaffinity assays are usually based on the use of long lifetime lanthanide chelates as the donor molecules and organic chromophores (nanosecond lifetime) as the acceptor molecules. The lifetime of these lanthanide chelates is typically in the range of ˜100-2500 μs depending on the chelate structure. The long lifetime of the donor (compared to the lifetime of free acceptor) allows the energy-transfer based acceptor signal to occur in the microsecond time region and this enables the time-resolved detection of the FRET based acceptor signal. Time-resolved detection (signal is integrated with certain delay time after the excitation pulse) in the microsecond time region eliminates efficiently the effect of nanosecond-lifetime background signal from the sample matrix and thus improves the signal/noise ratio (S/B-ratio) of the assay. Other benefit of lanthanide donors is that the emission spectrum of lanthanides consists of narrow peaks, which enables the spectral reduction (with optical filtering) of donor background in acceptor emission measurement channel. When measurement wavelength is selected suitably this allows additional improvement to S/B-ratio in acceptor emission based TR-FRET assays.
The ability to adjust the lifetime of the energy transfer based acceptor fluorescence by changing the energy transfer rate is not fully utilised in TR-FRET measurements and applications. Neither the ability to shorten the lifetime of energy transfer signal to a few microseconds when using ms-lifetime donor has been utilised in purpose to increase the TR-FRET assay sensitivity. For example the acceptor lifetime adjustment have been adapted in an application, in which so-called lifetime and colour tailored fluorophores have been developed (Chen and Selvin (2000) J. Am. Chem. Soc., 122, 657-660). In this application ms-lifetime donor and ns-lifetime acceptor are incorporated into a rigid template and the formed energy transfer complex with certain energy transfer efficiency is considered as a new single fluorophore, which can be further attached to molecule of interest. However, the acceptor lifetime adjustment is not used directly to optimise the TR-FRET measurement. In some published articles very short lifetime fluorescence signals (microsecond time scale) have been obtained in the acceptor measurement channel, when using millisecond lifetime donors and ns-lifetime acceptors, but the shortness of the signal has not been especially utilised in purpose to improve the assay sensitivity. In some particular cases the short lifetime acceptor population has been discarded during the TR data analysis based on the assumption that the short lifetime signal is caused by directly excited acceptor molecules or by a detector artifact (For example, see U.S. Pat. No. 5,656,433, Selvin et al (1994) J. Am. Chem Soc., 116, 6029-6030, Selvin et al (1994) Proc. Natl. Acad. Sci USA, 91, 10024-10028). Nevertheless directly excited ns-lifetime acceptor can not emit significantly in microsecond time scale due to very strong signal attenuation as a function of time. In addition to a detector artifact, also a high efficiency energy transfer process can produce very strong signal, which have a lifetime of few microseconds. With sophisticated TR instrument this type of phenomenon can be used to improve the sensitivity of TR-FRET assay.
There is a great need for TR-FRET assays with increased sensitivity for use in the detection of a single analyte, but particularly for use in multianalyte assays. The aim of the present invention is to overcome the limitations mentioned above.